A hybridoma cell line that secretes monoclonal antibodies against BHT and its analogues

Abstract

This invention relates to a hybridoma cell line that secretes monoclonal antibodies against BHT and its analogues, belonging to the field of immunoassay technology. The monoclonal antibodies secreted by the hybridoma cell line of this invention exhibit good sensitivity to BHT and its analogues. The monoclonal antibodies of this invention can simultaneously detect BHT, BHA, and TBHQ, and have an IC50 value for BHT. 50 The concentration was 83.08 ng / mL, and the IC50 for BHA was... 50 The concentration was 92.2 ng / mL, and the IC50 for TBHQ was [missing value]. 50 With a concentration of 86.0 ng / mL, the monoclonal antibody of this invention can simultaneously detect trace amounts of BHT, BHA, and TBHQ, and can be used for the residual detection of BHT, BHA, and TBHQ.

Description

Technical Field

[0001] This invention relates to the field of immunoassay technology, and in particular to a hybridoma cell line that secretes monoclonal antibodies against BHT and its analogues. Background Technology

[0002] Common synthetic antioxidants include BHT, BHA, and TBHQ. BHT, or Butylated hydroxytoluene, is chemically known as 2,6-di-tert-butyl-p-cresol. Its mechanism of action involves providing hydrogen atoms to react with free radicals, interrupting the chain reaction of oxidation and thus preventing or delaying oxidation. Specifically, BHT can provide a hydrogen atom to a peroxide radical, transforming it into a peroxide, while BHT itself becomes a stable phenoxy radical. These two phenoxy radicals can react to generate BHT and diene compounds, thus regenerating BHT. BHA, also known as tert-butyl-4-hydroxyanisole, is classified as a Group 2B carcinogen. TBHQ, also known as tert-butylhydroquinone, is carcinogenic. BHT and its analogues are commonly used as food additives to delay food rancidity and maintain its color, aroma, and texture. In addition, it is widely used in cosmetics, the plastics and rubber industries, and petroleum products. However, excessive amounts of BHT and its analogues are carcinogenic, therefore precise detection is required.

[0003] Currently, the detection methods for BHT and its analogues are mainly instrumental, with gas chromatography, liquid chromatography, and gas chromatography-mass spectrometry being commonly used. Although these chromatographic methods offer high sensitivity and specificity, they also have drawbacks, such as the need for thorough sample purification, high solvent consumption, expensive equipment, and skilled technicians. Therefore, a rapid and simple method for detecting the content of BHT and its analogues is needed.

[0004] Enzyme-linked immunosorbent assay (ELISA) is an extremely efficient, sensitive, and rapid detection method. It requires simple sample pretreatment, involves few purification steps, has a large analytical capacity, low detection cost, and is easy to operate, making it suitable for rapid on-site detection of large numbers of samples. Therefore, it is widely used in drug residue analysis. However, the prerequisite for using ELISA to detect BHT and its analogues is obtaining monoclonal antibodies capable of simultaneously detecting BHT and its analogues. Therefore, finding a method for preparing a monoclonal antibody capable of simultaneously detecting BHT and its analogues is crucial. Currently, there is a lack of monoclonal antibodies capable of simultaneously detecting BHT and its analogues in existing technologies. Summary of the Invention

[0005] Therefore, the technical problem to be solved by the present invention is to overcome the lack of a monoclonal antibody in the prior art that can simultaneously detect BHT and its analogues.

[0006] To address the aforementioned technical problems, this invention provides a hybridoma cell line that secretes monoclonal antibodies against BHT and its analogues. The monoclonal antibodies secreted by this hybridoma cell line can simultaneously detect BHT and its analogues, and the IC50 of BHT... 50 The concentration was 83.08 ng / mL, and the IC50 for BHA was... 50 The concentration was 92.2 ng / mL, and the IC50 for TBHQ was [missing value]. 50 With a concentration of 86.0 ng / mL, the monoclonal antibody of this invention can simultaneously detect BHT and its analogues, and can be used for the residual detection of BHT, BHA and TBHQ.

[0007] The first objective of this invention is to provide a hybridoma cell line that was deposited on April 17, 2025, at the China General Microbiological Culture Collection Center (CGMCC), classified as a monoclonal cell line, with the deposit address at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, and the accession number CGMCC No. 46509.

[0008] Furthermore, the hybridoma cell line is obtained by immunizing animals with a complete antigen prepared from a hapten, wherein the structural formula of the hapten is shown in Formula I:

[0009]

[0010] Furthermore, the complete antigen is obtained from the hapten coupled with a carrier protein.

[0011] Furthermore, the carrier protein includes keyhole hemocyanin.

[0012] A second objective of this invention is to provide an application of the above-described hybridoma cell line in the detection of BHT and its analogues.

[0013] Furthermore, BHT and its analogues include BHT, BHA, and TBHQ.

[0014] A third objective of this invention is to provide a monoclonal antibody secreted by the aforementioned hybridoma cell line.

[0015] A fourth objective of this invention is to provide an application of the above-described monoclonal antibody in the detection of BHT and its analogues.

[0016] A fifth objective of this invention is to provide a detection product for BHT and its analogues, the detection product comprising the aforementioned monoclonal antibody.

[0017] Furthermore, the test product also includes a coating agent.

[0018] Furthermore, the coating is prepared from a hapten-conjugated carrier protein, wherein the carrier protein includes chicken ovalbumin.

[0019] The beneficial effects of this invention are:

[0020] This invention provides a hybridoma cell line that secretes monoclonal antibodies against BHT and its analogues. The monoclonal antibodies secreted by this hybridoma cell line can simultaneously detect BHT and its analogues, and the IC50 for BHT is [not specified]. 50 The concentration was 83.08 ng / mL, and the IC50 for BHA was... 50 The concentration was 92.2 ng / mL, and the IC50 for TBHQ was [missing value]. 50 With a concentration of 86.0 ng / mL, the monoclonal antibody of this invention can simultaneously detect BHT and its analogues, and can be used for the residual detection of BHT, BHA and TBHQ.

[0021] Preservation of biological materials

[0022] The monoclonal cell line EBF was deposited on April 17, 2025, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 46509, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. Attached Figure Description

[0023] To make the content of this invention easier to understand, the invention will be further described in detail below with reference to specific embodiments and accompanying drawings, wherein...

[0024] Figure 1 This is a standard curve showing the inhibition of BHT, BHA, and TBHQ by monoclonal antibodies of BHT and its analogues according to the present invention. Detailed Implementation

[0025] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can better understand and implement the present invention. However, the embodiments described are not intended to limit the present invention.

[0026] The culture media involved in the following examples are as follows:

[0027] RPMI-1640 medium (mg / L): L-arginine 290, L-asparagine 50, L-aspartic acid 20, L-cysteine ​​dihydrochloride 65.15, L-glutamic acid 20, glycine 10, L-histidine 15, L-hydroxyproline 20, L-isoleucine 50, L-leucine 50, L-lysine hydrochloride 40, L-methionine 15, L-phenylalanine 15, L-proline 20, L-serine 30, L-threonine 20, L-tryptophan 5. L-Tyrosine 23.19, L-Valine 20, Para-aminobenzoic acid 1, Calcium nitrate 100, Anhydrous magnesium sulfate 48.84, Anhydrous sodium dihydrogen phosphate 676.13, Potassium chloride 400, Sodium chloride 6000, Glucose 2000, Reduced glutathione 1, Phenol red 5, L-Glutamine 300, Biotin 0.2, D-Calcium pantothenate 0.25, Folic acid 1, I-Inositol 35, Nicotinamide 1, Choline chloride 3, Pyridoxine hydrochloride 1, Riboflavin 0.2, Thiamine hydrochloride 1, Vitamin B12 0.005, Sodium bicarbonate 2000.

[0028] The reagents involved in the following examples are as follows:

[0029] Carbonate buffer (CBS): Weigh 1.59g of Na2CO3 and 2.93g of NaHCO3, dissolve them separately in a small amount of double-distilled water and mix them together. Add double-distilled water to about 800mL and mix well. Adjust the pH to 9.6 and add double-distilled water to a final volume of 1000mL. Store at 4℃ for later use.

[0030] Phosphate-buffered saline (PBS): 8.00g NaCl, 0.2g KCl, 0.2g KH2PO4, 2.9g Na2HPO4·12H2O, dissolved in 800mL pure water, pH adjusted to 7.2-7.4 with NaOH or HCl, and then brought to a final volume of 1000mL.

[0031] PBST: PBS containing 0.05% Tween 20;

[0032] Antibody dilution solution: PBS containing 0.1% gelatin;

[0033] TMB colorimetric solution: Solution A: 18.43g Na2HPO4·12H2O, 9.33g citric acid, diluted to 1000mL with pure water; Solution B: 60mg TMB dissolved in 100mL ethylene glycol. Mix solutions A and B in a 5:1 ratio to obtain the TMB colorimetric solution. Mix again before use.

[0034] The detection methods involved in the following embodiments are as follows:

[0035] Method for detecting the inhibition rate of BHT compounds: The optimal antigen and antibody concentrations for ic-ELISA were selected using a checkerboard assay. The antigen was diluted to 0.01, 0.03, 0.1, and 0.3 μg / mL with carbonate buffer (CBS), and the antibody was diluted to 0.03, 0.1, 0.3, and 1 μg / mL with antibody dilution buffer. After selecting the optimal operating point, the BHT compound standard was diluted to eight concentrations (0, 12.5, 25, 50, 100, 200, 400, and 800 ng / mL). Following the ic-ELISA procedure, the results were plotted using OriginPro 8.5 (see results below). Figure 1 As shown), standard inhibition curves for BHT-type compounds were obtained, and IC50 was calculated. 50 .

[0036] Example 1: Synthesis of complete antigens of BHT and its analogues

[0037] 3-(3,5-di-tert-butyl-4-hydroxyphenyl)propionic acid was selected as the hapten. 7.5 mg of 3-(3,5-di-tert-butyl-4-hydroxyphenyl)propionic acid and 6.2 mg of N-hydroxysuccinimide (NHS) were weighed and dissolved in 200 μL of N,N-dimethylformamide (DMF). The mixture was stirred at room temperature for 10 min to obtain the hapten solution. Then, 10.35 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) was weighed and added to the above hapten solution. The mixture was stirred at room temperature for 6-8 h to activate the hapten. 6 mg of keyhole hemocyanin (KLH) was added to 3 mL of 0.01 M carbonate buffer (CBS) and dissolved thoroughly. The activated hapten was then slowly added to the diluted KLH solution and stirred overnight at room temperature. Then, the antigen was dialyzed with 0.01M PBS solution to remove unreacted small molecules, resulting in a relatively pure complete antigen, which was then identified by ultraviolet absorption scanning.

[0038] Example 2: Synthesis of BHT and its analogues coated with antigens

[0039] 3.34 mg of 3-(3,5-di-tert-butyl-4-hydroxyphenyl)propionic acid and 2.75 mg of N-hydroxysuccinimide (NHS) were dissolved in 200 μL of anhydrous N,N-dimethylformamide (DMF) and reacted with the solution at room temperature for 10 min. 4.6 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) was dissolved in the above solution and reacted with the solution at room temperature for 6-8 h to obtain the hapten activation solution. 6 mg of chicken ovalbumin (OVA) was dissolved in carbonate buffer (CBS). The hapten activation solution was slowly added to the protein dilution solution and stirred overnight at room temperature. Then, the reaction solution was dialyzed against 0.01 M PBS to remove unreacted small molecules, yielding the coating antigen.

[0040] Example 3: Preparation of hybridoma cell lines secreting monoclonal antibodies against BHT and its analogues

[0041] 1. Acquisition of immunity in animals

[0042] BHT and its analogue complete antigens were emulsified with an equal amount of Freund's adjuvant and then administered to BALB / c mice via subcutaneous injection at multiple sites on the back of the neck (except for sprint immunization). The initial immunization used complete Freund's adjuvant at a dose of 100 μg / mouse. For subsequent booster immunizations, incomplete Freund's adjuvant was used at half the dose (50 μg / mouse). For sprint immunizations, no adjuvant was used; the adjuvant was diluted directly with physiological saline and injected intraperitoneally at a dose halved (25 μg / mouse). The interval between the initial and second booster immunizations was one month, the interval between multiple booster immunizations was 21 days, and the interval between sprint immunizations and the final booster immunization was 18-21 days. The immunization effect in mice was observed using an indirect competitive enzyme-linked immunosorbent assay (ic-ELISA), specifically by detecting the serum titer and inhibition.

[0043] 2. Cell fusion

[0044] Three days after the sprint immunization, cell fusion was performed using the standard PEG (polyethylene glycol, molecular weight 4000) method, with the following specific steps:

[0045] a. After euthanizing the mouse by tail dislocation and cervical dislocation, immediately disinfect the mouse in 75% alcohol for about 5 minutes. Under aseptic conditions, remove the spleen and grind it moderately with the rubber tip of a syringe and pass it through a 200-mesh cell sieve to obtain a spleen cell suspension. Collect the suspension, centrifuge (1200 rpm, 8 minutes), wash the spleen cells three times with RPMI-1640 medium, and after the last centrifugation, dilute the spleen cells to a certain volume, count them, and set them aside for later use.

[0046] b. Collection of SP2 / 0 cells: 7-10 days before fusion, SP2 / 0 tumor cells are expanded in RPMI-1640 medium containing 10% FBS (fetal bovine serum) in a 5% CO2 incubator. The number of SP2 / 0 tumor cells should reach 1-4 × 10⁶ cells / year before fusion. 7 To ensure that SP2 / 0 tumor cells are in the logarithmic growth phase before fusion, tumor cells are collected and suspended in RPMI-1640 basal culture medium for cell counting during fusion.

[0047] c. Fusion process (7 min): At min 1, add 1 mL of PEG 4000 to the cells dropwise, gradually increasing the speed; at min 2, allow to stand; at min 3 and min 4, add 1 mL of RPMI-1640 medium dropwise over 1 min; at min 5 and min 6, add 2 mL of RPMI-1640 medium dropwise over 1 min; at min 7, add 1 mL of RPMI-1640 medium dropwise every 10 s. Except for min 2, continuously agitate the solution. Then incubate at 37°C for 5 min; centrifuge (800 rpm, 8 min), discard the supernatant, and resuspend in RPMI-1640 selection medium containing 20% ​​fetal bovine serum and 2% 50×HAT. Add 200 μL / well to a 96-well cell plate and incubate at 37°C in a 5% CO2 incubator.

[0048] 3. Cell selection and cell line establishment

[0049] On day 3 after cell fusion, the fused cells were partially replaced with RPMI-1640 selection medium. On day 5, the medium was completely replaced with RPMI-1640 transition medium containing 20% ​​fetal bovine serum and 1% 100×HT. On day 7, the cell supernatant was collected for screening.

[0050] The screening process consists of two steps: First, positive cell wells are selected using ic-ELISA. Second, BHT and its analogues are used as standards, and the inhibitory effect on positive cells is determined using ic-ELISA.

[0051] Cell wells that showed good inhibition of BHT and its analogue standards were selected, and subcloning was performed using the limiting dilution method. The cells were then tested using the same method after seven days.

[0052] At least three subclonings were performed using the method described above to finally obtain hybridoma cell lines that secrete monoclonal antibodies against BHT and its analogues.

[0053] Example 4: Preparation and Identification of Monoclonal Antibodies Against BHT and its Analogs

[0054] 8-10 week old BALB / c mice were injected intraperitoneally with 1 mL of sterile paraffin oil; 7 days later, each mouse was injected intraperitoneally with 1×10 6 Hybridoma cells were used, and ascites fluid was collected starting from day seven. The ascites fluid was then purified for antibody treatment using the caprylic acid-saturated ammonium sulfate method.

[0055] Under slightly acidic conditions, octanoic acid can precipitate other proteins in the ascites fluid besides IgG immunoglobulin. After centrifugation, the precipitate is discarded. Then, an equal volume of saturated ammonium sulfate solution is used to precipitate IgG-type monoclonal antibodies. After centrifugation, the supernatant is discarded. The antibodies are dissolved in 0.01M PBS solution (pH 7.4), dialyzed to desalt, and finally purified monoclonal antibodies are obtained and stored at -20℃.

[0056] The IC50 of BHT and its analogue monoclonal antibodies against BHT was determined using an indirect competitive ELISA. 50 The value was 83.08 ng / mL, and the IC50 for BHA was... 50 The concentration was 92.2 ng / mL, and the IC50 for TBHQ was [missing value]. 50 The concentration was 86.0 ng / mL, indicating that it can simultaneously detect BHT, BHA and TBHQ, and has good sensitivity for BHT and its analogues, making it suitable for immunoassay detection of BHT and its analogues.

[0057] Example 5: Application of monoclonal antibodies against BHT and its analogues

[0058] Monoclonal antibodies prepared from hybridoma cell lines via in vivo ascites fluid were used in an ELISA assay for BHT and its analogues. The specific steps are as follows:

[0059] Coat 96-well microplates with 0.1 μg / mL of the coating stock diluted with carbonate buffer (CBS), 100 μL per well. After drying at 37°C for 2 h, wash the plates three times with PBST washing buffer, 200 μL per well each time, for 3 min each time, and pat dry.

[0060] Block with CBS containing 0.2% gelatin, 200 μL per well, dry at 37°C for 2 h, wash three times with PBST washing solution, 200 μL per well each time, 3 min each time, and pat dry.

[0061] BHT compound standard solutions of 0, 12.5, 25, 50, 100, 200, 400 and 800 ng / mL were prepared using phosphate-buffered saline (PBS). The standard solutions and the extracts of the samples to be tested were added to the pre-blocked ELISA plates, 50 μL per well, with each sample repeated in 3 wells. Then, 50 μL of BHT compound monoclonal antibody diluted to 0.1 μg / mL was added to each well. After incubation at 37°C for 30 min, the plates were washed and dried.

[0062] Add 100 μL of HRP-labeled goat anti-mouse IgG secondary antibody diluted 1:3000 with PBS containing 0.1% gelatin to each well, react at 37°C for 30 min, then wash and blot dry.

[0063] Add 100 μL of TMB colorimetric solution to each well, develop at 37℃ for 15 min, then add 50 μL of 2M H2SO4 stop solution to each well, and measure the absorbance at 450 nm.

[0064] Add recycling and sample pretreatment:

[0065] Vegetable oil was selected as the test sample.

[0066] Weigh out three 5g portions of the sample to be tested, and add 20ppb, 100ppb, and 500ppb of a mixed standard of BHT, BHA, and TBHQ (1:1:1) to each sample respectively (based on the antibody linear range and IC50). 50 (Set the desired concentration), add 5 mL of methanol, vortex at high speed for 5 min, and centrifuge at 5000g for 5 min. Extract the supernatant repeatedly with methanol five times, combine the extracts from each extraction, and dry the extract at 40℃ to a final volume of 0.5 mL. Reconstitute the concentrate with 4.5 mL of PBS solution containing 10% methanol. Recovery assays were performed using an indirect competitive ELISA, with recoveries of 82%, 113%, and 116%, respectively.

[0067] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A hybridoma cell line, characterized in that, The hybridoma cell line was named a monoclonal cell line and its accession number was CGMCC No. 46509.

2. The use of the hybridoma cell line according to claim 1 in the detection of BHT, BHA and / or TBHQ.

3. A monoclonal antibody secreted by the hybridoma cell line of claim 1.

4. The use of the monoclonal antibody according to claim 3 in the detection of BHT, BHA and / or TBHQ.

5. A detection product for BHT and its analogues, characterized in that, The detection product includes the monoclonal antibody as described in claim 3, and the BHT and its analogues are BHT, BHA and TBHQ.

6. The testing product according to claim 5, characterized in that, The tested products also include coating agents.

7. The testing product according to claim 6, characterized in that, The coating is prepared from a hapten-coupled carrier protein, wherein the carrier protein includes chicken ovalbumin.

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